Selective Immobilization of S4 in Domain III and IV of Rat Brain Nav1.2 Shown by Omega Currents

Abstract

Recently, we have identified the minimally necessary number of mutations of the outer positively charged arginine (R) or lysine (K) to glutamine (Q) in the voltage sensor S4 of each domain, producing an inward omega leak current in the resting state. In all four domains a double gap (RRn,n+1QQ) gave distinct omega currents. In this study we use these double gap channel constructs as a tool to investigate which S4 of the four domains I-IV is immobilized by inactivation. The recovery time constant of sodium current after inactivation was measured with a classical double pulse protocol for a wide range of recovery potentials from −100 to −240 mV. In addition, the onset of the omega current at the same recovery potentials was measured twofold: without and with an inactivating prepulse. We found that the onset of omega current was fast and not affected by inactivation in domain I and II; however, in domain III and IV the onset was fast without prepulse but was slowed after the inactivating prepulse. The return to the resting state seems to be hindered due to immobilization. The time constant of the recovery of omega current matches well the recovery of sodium current over the wide potential range studied. We corroborated our results by using the mutation R4H in S4DIV, which slows the sodium current recovery about twentyfold (Kühn and Greeff, 1999). Adding the mutation R4H in S4DIV to our double gap constructs, we found that the omega current was also slowed by the same factor. This suggests that the same mechanism which keeps the alpha pore closed for ionic current in inactivated channels would also hinder the return of S4III and S4IV to the resting position.